We propose to continue our study of transdermal delivery mechanisms with electric pulses. Our long-range goal is to develop convenient, safe and effective methods that can be applied to introduce drugs and vaccines across the skin barrier, for the purpose of controlled release, and topical treatment of local ailments including skin cancer. The stratum corneum (SC), the top layer of the skin, is the major barrier to transdermal transport, and contributes most of the electrical resistance of the skin. Electroporation of the SC can cause a breakdown of this barrier leading to an enhanced transdermal transport by several orders of magnitude. During the past grant period, we have determined the sequence of structural/functional events following the pulse application, leading to a transient permeabilization of the skin. One of our major discoveries is the transport enhancing effect of anionic lipids when applied with electroporation. We propose: (1) To determine the mechanism by which anionic lipids enhance electropermeabilization of the skin-we will test our hypothesis that exogenous anionic lipids mix with SC lipids and form local foci that are susceptible to electropermeabilization. We will then apply our knowledge of the mechanism to maximize the benefit of lipid enhancers. (2) To test the hypothesis that transdermal transport of charged molecules by electroporation is mainly via electrophoresis through hydrophilic electropores (channels)-we will compare the transport of a series of model peptides of varying charge/mass ratio and hydrophobicity. The study will be extended too examine the transport of four insulin analogs. (3) To apply our new-found knowledge to test for facilitated transdermal transport of photodynamic therapy (PDT) drugs through excised porcine skin, live murine skin, and human skin biopsies -ALA, Photofrin and HPPH will be delivered transdermally using our newly developed protocols, and their photodynamical therapeutic effect on colo26 tumor-bearing mice will be evaluated. Once the physicochemical mechanisms of skin permeabilization are known, novel, efficient, minimally invasive protocols can be designed for transdermal drug and vaccine delivery and tissue fluid monitoring.